Identification, quantification, and characterization of intracellular molecules in live cells are essential to dissect the intracellular pathways and networks to understand physiology and pathogenesis at the cellular level (1-5). Cell lysis by disrupting the cellular membrane to release intracellular molecules is a conventional laboratory technique to prepare samples for analysis of genes, proteins, and metabolites (6-8). Due to the termination of cell lives that results from this procedure, the progressive information is lost. The inconsistency of molecular background in the cell preparations for samples taken at different points in time largely compromises the study of cell differentiation, pathogenesis development, and therapeutic effectiveness. The extraction of intracellular molecules without killing cells so that repetitive sampling can be conducted at successive points in time is becoming an imperative and urgent mission.
Additionally, cellular heterogeneity is frequently observed, particularly in cancer cells (9). However, the traditional biochemical analysis only provides the average of the cellular information with an ensemble of molecules from a large quantity of cells. Single-cell analysis is essential to obtain the physiological and pathological characteristics with respect to the genetic, proteomic, spatial, and temporal diversity of cells in cell biology and cancer research (10-12). Although microfluidics and lab-on-chip have been widely applied to single-cell manipulation via cell trapping, isolation, and sorting, the analyte extraction still relies on complete lysis (13, 14).
Physical penetration of the cell membrane has exhibited low invasiveness in the extraction or release of intracellular molecules (15, 16). Nanoneedle and optoporation were utilized for subcellular disruption and manipulation in living cells, but special and sophisticated setups are often required to wage the high spatial resolution and precise manipulation (17-20). Electroporation was also demonstrated to release intracellular proteins without loss of cell viability (21). However, the efficiency can be limited due to its dependence on diffusion to release the molecules. To date, the efficient extraction of molecules from live cells at the single cell level remains a significant challenge in biotechnology.
Extraction of intracellular molecules is crucial to the study of cellular signaling pathways. Disruption of the cellular membrane remains the established method to release the intracellular contents, which inevitably terminates the time course of biological processes. Also, conventional lab extractions mostly employ bulky materials that ignore the heterogeneity of each cell.
Current technical barriers in molecular sampling compromise the biomedical research regarding the diversity of cellular background. Usually hundreds and thousands of cells are lysed to release their contents. As such, the differences among individual cells are averaged out. The progressive cellular information can only be obtained by analysis of cells terminated at sequential points in time, or by using external fluorescent and chemical labels that may interfere with pathways. As such, there exists a need for improved methods of interrogating cellular signaling pathways.